RU2019111616A - METHODS AND COMPOSITIONS FOR DIRECTED GENOMA MODIFICATION - Google Patents

METHODS AND COMPOSITIONS FOR DIRECTED GENOMA MODIFICATION Download PDF

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RU2019111616A
RU2019111616A RU2019111616A RU2019111616A RU2019111616A RU 2019111616 A RU2019111616 A RU 2019111616A RU 2019111616 A RU2019111616 A RU 2019111616A RU 2019111616 A RU2019111616 A RU 2019111616A RU 2019111616 A RU2019111616 A RU 2019111616A
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interest
genomic locus
locus
genetic modification
nucleotide
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RU2725520C2 (en
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Дэвид ФРЕНДЕВЕЙ
Войтек АУЕРБАХ
Ка-Ман Венус ЛАЙ
Джунко КУНО
Дэвид М. ВАЛЕНЦУЕЛА
Джордж Д. Янкопулос
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Регенерон Фармасьютикалс, Инк.
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Claims (107)

1. Способ in vitro модификации генома в представляющем интерес геномном локусе в нечеловеческой плюрипотентной клетке млекопитающего, включающий внесение в плюрипотентную клетку1. Method for in vitro modification of the genome in a genomic locus of interest in a non-human pluripotent mammalian cell, comprising insertion into a pluripotent cell (a) белка Cas9, матричной РНК (мРНК), кодирующей белок Cas9, или ДНК, кодирующей белок Cas9;(a) Cas9 protein, messenger RNA (mRNA) that encodes Cas9 protein, or DNA that encodes Cas9 protein; (b) направляющей РНК, содержащей РНК CRISPR, которая гибридизируется с целевой последовательностью CRISPR в представляющем интерес геномном локусе и tracrРНК или ДНК, кодирующей направляющую РНК;(b) a guide RNA containing CRISPR RNA, which hybridizes with the target CRISPR sequence in the genomic locus of interest and the tracrRNA or DNA that encodes the guide RNA; (c) крупного направляющего вектора (LTVEC), который составляет по меньшей мере 10 т.п.о. и содержит нуклеотидную вставку, фланкируемую:(c) a large guide vector (LTVEC) that is at least 10 kb. and contains a nucleotide insert flanked by: (i) 5’ плечом гомологии, которое гомологично 5’ целевой последовательности в представляющем интерес геномном локусе, где 5’ плечо гомологии составляет от около 5 т.п.о. до около 200 т.п.о., и(i) a 5 ’shoulder of homology, which is homologous to a 5’ target sequence in a genomic locus of interest, where 5 ’shoulder of homology is from about 5 kb. up to about 200 kbp and (ii) 3’ плечом гомологии, которое гомологично 3’ целевой последовательности в представляющем интерес геномном локусе, где 3’ плечо гомологии составляет от около 5 т.п.о. до около 200 т.п.о., (ii) a 3 ’shoulder of homology, which is homologous to a 3’ target sequence in a genomic locus of interest, where 3 ’shoulder of homology is from about 5 kb. up to about 200 kb, при этом направляющую РНК конструируют, чтобы избежать распознавания любой последовательности в нуклеотидной вставке, и при этом после внесения в плюрипотентную клетку (a) белка Cas9, мРНК, кодирующей белок Cas9, или ДНК, кодирующей белок Cas9, (b) направляющей РНК или ДНК, кодирующей направляющую РНК и (c) LTVEC происходит модификация генома плюрипотентной клетки для содержания направленной генетической модификации, включающей удаление области представляющего интерес геномного локуса, где делеция составляет от около 30 т.п.о. до около 200 т.п.о. и/или введение нуклеотидной вставки в представляющий интерес геномный локус, где вставка составляет от около 30 т.п.о. до около 200 т.п.о.,however, the guide RNA is designed to avoid recognizing any sequence in the nucleotide insert, and after inserting (a) Cas9 protein, mRNA that encodes the Cas9 protein, or DNA that encodes the Cas9 protein, (b) directing the RNA or DNA into the pluripotent cell, the coding guide RNA and (c) LTVEC, modifies the genome of the pluripotent cell to contain a directed genetic modification, including the removal of a region of interest of the genomic locus, where the deletion is from about 30 kb. up to about 200 kb and / or introducing a nucleotide insert into a genomic locus of interest, where the insert is from about 30 kb. up to about 200 kb, при этом если направленная генетическая модификация включает введение нуклеотидной вставки в представляющий интерес геномный локус, где вставка составляет от около 30 т.п.о. до около 200 т.п.о., тогда LTVEC составляет по меньшей мере 40 т.п.о.moreover, if the directed genetic modification includes the introduction of a nucleotide insert into a genomic locus of interest, where the insert is from about 30 kb. to about 200 kbp, then the LTVEC is at least 40 kbp. 2. Способ по п. 1, где направленная генетическая модификация содержит делецию области представляющего интерес геномного локуса и направляющую РНК конструируют для создания двухцепочечного разрыва в области представляющего интерес геномного локуса, предназначенного для удаления.2. The method of claim 1, wherein the directed genetic modification comprises a deletion of the region of interest of the genomic locus and the guide RNA is designed to create a double-stranded break in the region of the genomic interest of interest for deletion. 3. Способ по п. 1 или 2, где целевая последовательность CRISPR находится в области 5’ конца, представляющего интерес геномного локуса, предназначенного для удаления.3. The method of claim 1 or 2, wherein the target CRISPR sequence is in the region of the 5 ′ end representing the interest of the genomic locus to be deleted. 4. Способ по п.3, где целевая последовательность CRISPR составляет от 50-1000 пар оснований от конечной точки делеции.4. The method of claim 3, wherein the target CRISPR sequence is from 50-1000 base pairs from the deletion end point. 5. Способ по п. 1 или 2, где целевая последовательность CRISPR находится в области 3’ конца, представляющего интерес геномного локуса, предназначенного для удаления.5. The method according to claim 1 or 2, where the target CRISPR sequence is in the region of the 3 'end, representing the interest of the genomic locus to be deleted. 6. Способ по п.5, где целевая последовательность CRISPR составляет от 50-1000 пар оснований от конечной точки делеции.6. The method according to claim 5, where the target CRISPR sequence is from 50-1000 base pairs from the end point of the deletion. 7. Способ по п. 1 или 2, где целевая последовательность CRISPR расположена в интроне, экзоне, промотере, энхансере, регуляторной области или любой не кодирующей белок области.7. The method according to claim 1 or 2, wherein the target CRISPR sequence is located in an intron, exon, promoter, enhancer, regulatory region, or any non-protein coding region. 8. Способ по п. 1 или 2, где целевая последовательность CRISPR расположена в пределах кодирующей области гена или в пределах регуляторных областей, которые влияют на экспрессию гена.8. The method according to claim 1 or 2, where the target CRISPR sequence is located within the coding region of the gene or within the regulatory regions that affect gene expression. 9. Способ по п. 1 или 2, где направляющая РНК содержит SEQ ID NO: 2, 3, 4, 5, 6, 7 или 8.9. The method according to claim 1 or 2, wherein the guide RNA comprises SEQ ID NO: 2, 3, 4, 5, 6, 7 or 8. 10. Способ по п. 9, где направляющая РНК содержит SEQ ID NO: 3, 4, 5 или 7.10. The method according to p. 9, where the guide RNA contains SEQ ID NO: 3, 4, 5 or 7. 11. Способ по п. 1 или 2, где РНК CRISPR и tracrРНК вносят вместе в одной молекуле нуклеиновой кислоты, содержащей РНК CRISPR и tracrРНК.11. A method according to claim 1 or 2, wherein the CRISPR RNA and tracrRNA are introduced together in a single nucleic acid molecule containing the CRISPR RNA and tracrRNA. 12. Способ по п.11, где одна молекула нуклеиновой кислоты содержит РНК CRISPR и tracrРНК слитые вместе с образованием одной направляющей РНК (онРНК)12. The method according to claim 11, where one nucleic acid molecule contains CRISPR RNA and tracrRNA fused together to form one directing RNA (onRNA) 13. Способ по п.1 или 2, где РНК CRISPR и tracrРНК вносят отдельно.13. The method according to claim 1 or 2, where CRISPR RNA and tracrRNA are introduced separately. 14. Способ по п. 1 или 2, где белок Cas9, РНК CRISPR и tracrРНК вносят в виде комплекса белок-РНК.14. The method according to claim 1 or 2, wherein the Cas9 protein, CRISPR RNA and tracrRNA are introduced as a protein-RNA complex. 15. Способ по п. 1 или 2, где15. The method according to claim 1 or 2, where (I)(a) ДНК, кодирующая белок Cas9, находится в форме первой экспрессионной конструкции, содержащей первый промотор, функционально связанный с первой нуклеиновой кислотой, кодирующей белок Cas9; (I) (a) DNA encoding the Cas9 protein is in the form of a first expression construct containing a first promoter that is functionally linked to the first nucleic acid encoding the Cas9 protein; (b) ДНК, кодирующая РНК CRISPR, находится в форме второй экспрессионной конструкции, содержащей второй промотор, функционально связанный со второй нуклеиновой кислотой, кодирующей РНК CRISPR; и(b) DNA encoding CRISPR RNA is in the form of a second expression construct containing a second promoter, functionally linked to a second nucleic acid encoding CRISPR RNA; and (c) ДНК, кодирующая tracrРНК, находится в форме третьей экспрессионной конструкции, содержащей третий промотор, функционально связанный с третьей нуклеиновой кислотой, кодирующей tracrРНК; (c) DNA encoding tracrRNA is in the form of a third expression construct containing a third promoter, functionally linked to a third nucleic acid encoding tracrRNA; при этом первый, второй и третий промоторы активны в плюрипотентной клетке, и где первая, вторая и третья экспрессионные конструкции находятся в одной молекуле нуклеиновой кислоты или в отдельных молекулах нуклеиновых кислот; илиwherein the first, second, and third promoters are active in the pluripotent cell, and where the first, second, and third expression constructs are located in a single nucleic acid molecule or in separate nucleic acid molecules; or (II)(a) ДНК, кодирующая белок Cas9, находится в форме первой экспрессионной конструкции, содержащей первый промотор, функционально связанный с первой нуклеиновой кислотой, кодирующей белок Cas9; и(Ii) (a) DNA encoding the Cas9 protein is in the form of a first expression construct containing a first promoter, functionally linked to the first nucleic acid encoding the Cas9 protein; and (b) ДНК, кодирующая РНК CRISPR, и ДНК, кодирующая tracrРНК, находятся в форме второй экспрессионной конструкции, содержащей второй промотор, функционально связанный со второй нуклеиновой кислотой, кодирующей направляющую РНК (нРНК), содержащую РНК CRISPR и tracrРНК; (b) DNA encoding CRISPR RNA and DNA encoding tracrRNA are in the form of a second expression construct containing a second promoter functionally linked to a second nucleic acid encoding a guide RNA (nRNA) containing CRISPR RNA and tracrRNA; при этом первый и второй промоторы активны в плюрипотентной клетке, и гдеwherein the first and second promoters are active in a pluripotent cell, and where первая и вторая экспрессионные конструкции находятся в одной молекуле нуклеиновой кислоты или в отдельных молекулах нуклеиновых кислот.The first and second expression constructs are located in the same nucleic acid molecule or in separate nucleic acid molecules. 16. Способ по п. 1 или 2, где направленная генетическая модификация включает одновременное удаление эндогенной нуклеотидной последовательности в представляющем интерес геномном локусе и введение нуклеотидной вставки в представляющий интерес геномный локус.16. The method according to claim 1 or 2, wherein the directed genetic modification involves the simultaneous removal of the endogenous nucleotide sequence in the genomic locus of interest and the introduction of a nucleotide insert in the genomic locus of interest. 17. Способ по п. 16, где удаленная эндогенная нуклеотидная последовательность составляет от 30 т.п.о. до около 110 т.п.о., а нуклеотидная вставка составляет от около 40 т.п.о. до около 140 т.п.о.17. The method of claim 16, wherein the remote endogenous nucleotide sequence is from 30 kb. to about 110 kbp, and the nucleotide insert is from about 40 kbp. up to about 140 kb 18. Способ по п. 1 или 2, где направленная генетическая модификация является биаллельной генетической модификацией.18. The method according to claim 1 or 2, wherein the directed genetic modification is a biallelic genetic modification. 19. Способ по п. 18, где 19. A method according to claim 18, where (a) биаллельная генетическая модификация включает удаление эндогенной нуклеотидной последовательности и введение нуклеотидной вставки в представляющий интерес геномный локус в двух гомологичных хромосомах; или(a) biallelic genetic modification involves removing the endogenous nucleotide sequence and introducing the nucleotide insert into the genomic locus of interest on two homologous chromosomes; or (b) модифицированная плюрипотентная клетка является компаунд-гетерозиготной или гемизиготной в представляющем интерес геномном локусе. (b) the modified pluripotent cell is a compound heterozygous or hemizygous in the genomic locus of interest. 20. Способ по п. 19, где направленная генетическая модификация в представляющем интерес геномном локусе в одной хромосоме включает удаление эндогенной нуклеотидной последовательности и введение нуклеотидной вставки.20. The method of claim 19, wherein the directed genetic modification in the genomic locus of interest in one chromosome involves removing the endogenous nucleotide sequence and introducing the nucleotide insert. 21. Способ по п. 20, где направленная генетическая модификация включает: (1) удаление эндогенной нуклеотидной последовательности в представляющем интерес геномном локусе в первой и второй гомологичных хромосомах; и (2) введение нуклеотидной вставки в представляющий интерес геномный локус в первой гомологичной хромосоме и разрушение представляющего интерес геномного локуса во второй гомологичной хромосоме.21. The method of claim 20, wherein the directed genetic modification comprises: (1) removing the endogenous nucleotide sequence at a genomic locus of interest in the first and second homologous chromosomes; and (2) inserting a nucleotide insert into a genomic locus of interest in the first homologous chromosome and disrupting the genomic locus of interest in the second homologous chromosome. 22. Способ по п. 1 или 2, где LTVEC составляет по меньшей мере 40 т.п.о., или где направленная генетическая модификация включает удаление области представляющего интерес геномного локуса при этом делеция составляет по меньшей мере 30 т.п.о. и LTVEC составляет по меньшей мере 15 т.п.о.22. The method according to claim 1 or 2, wherein the LTVEC is at least 40 kbp, or where the directed genetic modification involves removing a region of the genomic locus of interest with a deletion of at least 30 kbp. and LTVEC is at least 15 kb. 23. Способ по п. 1 или 2, где направленная генетическая модификация включает введение нуклеотидной вставки в представляющий интерес геномный локус, где нуклеотидная вставка составляет по меньшей мере 40 т.п.о.; или23. The method of claim 1 or 2, wherein the directed genetic modification comprises introducing a nucleotide insert into the genomic locus of interest, where the nucleotide insert is at least 40 kb; or где направленная генетическая модификация включает удаление области представляющего интерес геномного локуса, при этом делеция составляет по меньшей мере 30 т.п.о. и введение нуклеотидной вставки в представляющий интерес геномный локус, при этом нуклеотидная вставка составляет по меньшей мере 10 т.п.о.where the targeted genetic modification involves the removal of a region of the genomic locus of interest, with a deletion of at least 30 kb. and introducing a nucleotide insert into a genomic locus of interest, with a nucleotide insert of at least 10 kb. 24. Способ по п. 1 или 2, где нуклеотидная вставка составляет от около 40 т.п.о. до около 140 т.п.о.24. The method according to p. 1 or 2, where the nucleotide insert is from about 40 T. p. up to about 140 kb 25. Способ по п. 1 или 2, где целевая последовательность CRISPR непосредственно фланкируется последовательностью мотива, прилегающего к протоспейсеру (PAM).25. The method according to claim 1 or 2, where the target CRISPR sequence is directly flanked by a sequence of motif adjacent to the protospecer (PAM). 26. Способ по п. 1 или 2, где 26. The method according to claim 1 or 2, where (a) 5’ и 3’ плечи гомологии LTVEC в общей сумме составляют от 10 т.п.о. до 150 т.п.о. или 30 т.п.о. до 150 т.п.о в длину и/или(a) 5 ’and 3’ shoulders of LTVEC homology total up to 10 kb. up to 150 kb or 30 kb. up to 150 kb in length and / or (b) LTVEC составляет от 100 т.п.о. до 300 т.п.о. в длину.(b) LTVEC is from 100 kb. up to 300 kb in length. 27. Способ по п. 1 или 2, где направленная генетическая модификация включает:27. The method according to claim 1 or 2, where the directed genetic modification includes: (a) замещение эндогенной нуклеотидной последовательности гомологичной или ортологичной нуклеотидной последовательностью;(a) the replacement of the endogenous nucleotide sequence with a homologous or orthologous nucleotide sequence; (b) удаление эндогенной нуклеотидной последовательности;(b) removing the endogenous nucleotide sequence; (c) удаление эндогенной нуклеотидной последовательности, (c) removing the endogenous nucleotide sequence, при этом диапазон удаления составляет от по меньшей мере 30 т.п.о. до около 3 м.п.о.;wherein the range of removal is from at least 30 kb. up to about 3 m. p. (d) вставку экзогенной нуклеотидной последовательности;(d) insertion of an exogenous nucleotide sequence; (e) вставку экзогенной нуклеотидной последовательности в диапазоне от по меньшей мере 30 т.п.о. до около 400 т.п.о.;(e) inserting an exogenous nucleotide sequence ranging from at least 30 kb. up to about 400 kb .; (f) вставку экзогенной нуклеотидной последовательности, содержащей гомологичную или ортологичную нуклеотидную последовательность;(f) insertion of an exogenous nucleotide sequence containing a homologous or orthologous nucleotide sequence; (g) вставку химерной нуклеотидной последовательности, содержащей человеческую и нечеловеческую нуклеотидную последовательность;(g) insertion of a chimeric nucleotide sequence containing a human and non-human nucleotide sequence; (h) вставку кондиционального аллеля, фланкируемого целевыми последовательностями сайт-специфической рекомбиназы; (h) insertion of an air conditioned allele flanked by target site-specific recombinase sequences; (i) вставку селективного маркера или репортерного гена, функционально связанного с промотором, активным в плюрипотентной клетке; или(i) insertion of a selective marker or reporter gene, functionally linked to a promoter active in a pluripotent cell; or (j) их комбинацию.(j) their combination. 28. Способ по п. 1 или 2, где направленная генетическая модификация включает удаление области представляющего интерес геномного локуса при этом делеция составляет по меньшей мере 40 т.п.о. или где направленная генетическая модификация дополнительно включает введение нуклеотидной вставки в представляющий интерес геномный локус, где вставка составляет по меньшей мере 30 т.п.о. и удаление области представляющего интерес геномного локуса, при этом делеция составляет по меньшей мере 10 т.п.о.28. The method according to claim 1 or 2, wherein the directed genetic modification comprises removing the region of the genomic locus of interest with a deletion of at least 40 kb. or where the directed genetic modification further comprises introducing the nucleotide insert into the genomic locus of interest, where the insert is at least 30 kb. and deletion of the region of the genomic locus of interest, with a deletion of at least 10 kb. 29. Способ по п. 1 или 2, где удаляемая область представляющего интерес геномного локуса составляет от около 30 т.п.о. до около 110 т.п.о.29. The method according to claim 1 or 2, wherein the region of interest of the genomic locus of interest is from about 30 kb. up to about 110 kb 30. Способ по п. 1 или 2, где направленная генетическая модификация включает удаление области представляющего интерес геномного локуса, при этом делеция составляет по меньшей мере 30 т.п.о. и введение нуклеотидной вставки в представляющий интерес геномный локус, где вставка составляет по меньшей мере 30 т.п.о. 30. The method of claim 1 or 2, wherein the directed genetic modification comprises removing a region of the genomic locus of interest, wherein the deletion is at least 30 kb. and introducing the nucleotide insert into the genomic locus of interest, where the insert is at least 30 kb. 31. Способ по п. 1 или 2, где представляющий интерес геномный локус является эндогенным локусом плюрипотентной клетки.31. The method of claim 1 or 2, wherein the genomic locus of interest is the endogenous pluripotent cell locus. 32. Способ по п. 1 или 2, где представляющий интерес геномный локус включает гетерологичный или экзогенный сегмент ДНК, который был интегрирован в геном плюрипотентной клетки.32. The method of claim 1 or 2, wherein the genomic locus of interest comprises a heterologous or exogenous DNA segment that has been integrated into the genome of a pluripotent cell. 33. Способ по пп. 1 или 2, где представляющий интерес геномный локус представляет собой локус иммуноглобулина.33. The method according to paragraphs. 1 or 2, where the genomic locus of interest is an immunoglobulin locus. 34. Способ по п. 33, где локус иммуноглобулина кодирует34. The method of claim 33, wherein the immunoglobulin locus encodes (a) аминокислотную последовательность вариабельной области тяжелой цепи иммуноглобулина млекопитающего; или(a) amino acid sequence of the variable region of the mammalian immunoglobulin heavy chain; or (b) аминокислотную последовательность вариабельной области легкой цепи иммуноглобулина млекопитающего.(b) the amino acid sequence of the immunoglobulin light chain variable region of a mammal. 35. Способ по п. 34, где локус иммуноглобулина включает35. The method of claim 34, wherein the immunoglobulin locus comprises (a) неперестроенную нуклеотидную последовательность вариабельной области λ и/или κ легкой цепи млекопитающего; или(a) the unreconstructed nucleotide sequence of the variable region λ and / or κ of the mammalian light chain; or (b) перестроенную нуклеотидную последовательность вариабельной области λ и/или κ легкой цепи млекопитающего.(b) a rebuilt nucleotide sequence of the variable region λ and / or κ of the mammalian light chain. 36. Способ по п. 1 или 2, где представляющий интерес геномный локус представляет собой локус рецептора Т-клетки.36. The method of claim 1 or 2, wherein the genomic locus of interest is the T-cell receptor locus. 37. Способ по п. 36, где локус рецептора Т-клетки представляет собой локус альфа-рецептора Т-клетки.37. The method of claim 36, wherein the T cell receptor locus is the alpha receptor T cell locus. 38. Способ по п. 1 или 2, где представляющий интерес геномный локус включает локус Il2rg, локус ApoE, локус Rag1, локус Rag2, оба локуса Rag1 и Rag2, локус Adamts5, локус Trpa1, локус Folh1, локус Erbb4, локус Lrp5, локус C5 (Hc), локус Ror1 или локус Dpp4.38. The method of claim 1 or 2, wherein the genomic locus of interest includes the Il2rg locus, the ApoE locus, the Rag1 locus, the Rag2 locus, both the Rag1 and Rag2 loci, the Adamts5 locus, the Trpa1 locus, the Folh1 locus, the Erbb4 locus, the Lrp5 locus, the locus C5 (Hc), Ror1 locus or Dpp4 locus. 39. Способ по пп. 1 или 2, где нуклеотидная вставка включает геномную нуклеотидную последовательность, которая кодирует аминокислотную последовательность вариабельной области тяжелой цепи иммуноглобулина человека. 39. The method according to paragraphs. 1 or 2, where the nucleotide insert includes a genomic nucleotide sequence that encodes the amino acid sequence of the variable region of the heavy chain of a human immunoglobulin. 40. Способ по п. 39, где нуклеотидная вставка включает40. The method of claim 39, wherein the nucleotide insert includes (a) один или более функциональных человеческих генных сегментов VH, включающих VH1-2, VH1-3, VH1-8, VH1-18, VH1-24, VH1-45, VH1-46, VH1-58, VH1-69, VH2-5, VH2-26, VH2-70, VH3-7, VH3-9, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-30-3, VH3-30-5, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66, VH3-72, VH3-73, VH3-74, VH4-4, VH4-28, VH4-30-1, VH4-30-2, VH4-30-4, VH4-31, VH4-34, VH4-39, VH4-59, VH4-61, VH5-51, VH6-1, VH7-4-1, VH7-81 или их комбинацию;(a) one or more functional human gene segments V H , including V H 1-2, V H 1-3, V H 1-8, V H 1-18, V H 1-24, V H 1-45, V H 1-46, V H 1-58, V H 1-69, V H 2-5, V H 2-26, V H 2-70, V H 3-7, V H 3-9, V H 3-11, V H 3-13, V H 3-15, V H 3-16, V H 3-20, V H 3-21, V H 3-23, V H 3-30, V H 3- 30-3, V H 3-30-5, V H 3-33, V H 3-35, V H 3-38, V H 3-43, V H 3-48, V H 3-49, V H 3-53, V H 3-64, V H 3-66, V H 3-72, V H 3-73, V H 3-74, V H 4-4, V H 4-28, V H 4- 30-1, V H 4-30-2, V H 4-30-4, V H 4-31, V H 4-34, V H 4-39, V H 4-59, V H 4-61, V H 5-51, V H 6-1, V H 7-4-1, V H 7-81, or a combination of these; (b) один или более функциональных человеческих генных сегментов D, включающих D1-1, D1-7, D1-14, D1-20, D1-26, D2-2, D2-8, D2-15, D2-21, D3-3, D3-9, D3-10, D3-16, D3-22, D4-4, D4-11, D4-17, D4-23, D5-12, D5-5, D5-18, D5-24, D6-6, D6-13, D6-19, D6-25, D7-27 или их комбинацию; или(b) one or more functional human gene segments D, including D1-1, D1-7, D1-14, D1-20, D1-26, D2-2, D2-8, D2-15, D2-21, D3 -3, D3-9, D3-10, D3-16, D3-22, D4-4, D4-11, D4-17, D4-23, D5-12, D5-5, D5-18, D5-24 , D6-6, D6-13, D6-19, D6-25, D7-27, or a combination thereof; or (c) один или более функциональных генных сегментов JH, включающих JH1, JH2, JH3, JH4, JH5, JH6 или их комбинацию.(c) one or more functional gene segments J H, J H 1 comprising, J H 2, J H 3, J H 4, J H 5, J H 6 or a combination thereof. 41. Способ по п. 1 или 2, где нуклеотидная вставка включает геномную нуклеотидную последовательность, которая кодирует аминокислотную последовательность вариабельной области легкой цепи иммуноглобулина человека. 41. The method of claim 1 or 2, wherein the nucleotide insert includes a genomic nucleotide sequence that encodes the amino acid sequence of the human immunoglobulin light chain variable region. 42. Способ по п. 41, где нуклеотидная вставка включает42. The method of claim 41, wherein the nucleotide insert includes (a) один или более человеческих генных сегментов Vκ, включающих Vκ4-1, Vκ5-2, Vκ 7-3, Vκ 2-4, Vκ1-5, Vκ1-6, Vκ3-7, Vκ1-8, Vκ1-9, Vκ2-10, Vκ3-11, Vκ1-12, Vκ1-13, Vκ2-14, Vκ3-15, Vκ1-16, Vκ1-17, Vκ2-18, Vκ2-19, Vκ3-20, Vκ6-21, Vκ1-22, Vκ1-23, Vκ2-24, Vκ3-25, Vκ2-26, Vκ1-27, Vκ2-28, Vκ2-29, Vκ2-30, Vκ3-31, Vκ1-32, Vκ1-33, Vκ3-34, Vκ1-35, Vκ2-36, Vκ1-37, Vκ2-38, Vκ1-39, Vκ2-40 или их комбинацию;(a) one or more human Vκ gene segments, including Vκ4-1, Vκ5-2, Vκ 7-3, Vκ 2-4, Vκ1-5, Vκ1-6, Vκ3-7, Vκ1-8, Vκ1-9, Vκ2-10, Vκ3-11, Vκ1-12, Vκ1-13, Vκ2-14, Vκ3-15, Vκ1-16, Vκ1-17, Vκ2-18, Vκ2-19, Vκ3-20, Vκ6-21, Vκ1- 22, Vκ1-23, Vκ2-24, Vκ3-25, Vκ2-26, Vκ1-27, Vκ2-28, Vκ2-29, Vκ2-30, Vκ3-31, Vκ1-32, Vκ1-33, Vκ3-34, Vκ1-35, Vκ2-36, Vκ1-37, Vκ2-38, Vκ1-39, Vκ2-40 or a combination thereof; (b) один или более человеческих генных сегментов Vλ, включающих Vλ3-1, Vλ4-3, Vλ2-8, Vλ3-9, Vλ3-10, Vλ2-11, Vλ3-12, Vλ2-14, Vλ3-16, Vλ2-18, Vλ3-19, Vλ3-21, Vλ3-22, Vλ2-23, Vλ3-25, Vλ3-27 или их комбинацию; или(b) one or more human gene segments Vλ, including Vλ3-1, Vλ4-3, Vλ2-8, Vλ3-9, Vλ3-10, Vλ2-11, Vλ3-12, Vλ2-14, Vλ3-16, Vλ2- 18, Vλ3-19, Vλ3-21, Vλ3-22, Vλ2-23, Vλ3-25, Vλ3-27, or a combination of these; or (c) один или более человеческих генных сегментов Jκ, включающих Jκ1, Jκ2, Jκ3, Jκ4, Jκ5 или их комбинацию.(c) one or more human Jκ gene segments, including Jκ1, Jκ2, Jκ3, Jκ4, Jκ5, or a combination thereof. 43. Способ по п. 1 или 2, где нуклеотидная вставка включает полинуклеотид, кодирующий по меньшей мере область рецептора Т-клетки человека.43. The method of claim 1 or 2, wherein the nucleotide insert comprises a polynucleotide encoding at least a region of the human T-cell receptor. 44. Способ по п. 43, где рецептор Т-клетки представляет собой рецептор Т-клетки альфа.44. The method of claim 43, wherein the T-cell receptor is an alpha T-cell receptor. 45. Способ по п. 1 или 2, где нуклеотидная вставка включает по меньшей мере один аллель заболевания.45. A method according to claim 1 or 2, wherein the nucleotide insert includes at least one allele of the disease. 46. Способ по п. 45, где нуклеотидная вставка включает по меньшей мере один аллель заболевания человека из человеческого гена.46. The method of claim 45, wherein the nucleotide insert includes at least one allele of a human disease from a human gene. 47. Способ по п. 1 или 2, где направленная генетическая модификация приводит к гуманизированному геномному локус, включающему: (a) вставку гомологичной или ортологичной человеческой нуклеотидной последовательности; (b) замещение эндогенной нуклеотидной последовательности гомологичной или ортологичной нуклеотидной последовательностью; или (c) их комбинацию. 47. The method of claim 1 or 2, wherein the directed genetic modification results in a humanized genomic locus comprising: (a) inserting a homologous or orthologous human nucleotide sequence; (b) replacing the endogenous nucleotide sequence with a homologous or orthologous nucleotide sequence; or (c) their combination. 48. Способ по п. 47, где направленная генетическая модификация включает замену эндогенной нуклеотидной последовательности гомологичной или ортологичной человеческой нуклеотидной последовательностью.48. The method of claim 47, wherein the directed genetic modification comprises replacing the endogenous nucleotide sequence with a homologous or orthologous human nucleotide sequence. 49. Способ по п. 48, где направленная генетическая модификация включает удаление области представляющего интерес геномного локуса, при этом делеция составляет по меньшей мере 30 т.п.о. и введение нуклеотидной вставки в представляющий интерес геномный локус, где вставка составляет по меньшей мере 30 т.п.о. 49. The method of claim 48, wherein the directed genetic modification involves removing a region of the genomic locus of interest, wherein the deletion is at least 30 kb. and introducing the nucleotide insert into the genomic locus of interest, where the insert is at least 30 kb. 50. Способ по п. 49, где удаленная область составляет от 30 т.п.о. до около 110 т.п.о., а нуклеотидная вставка составляет от около 40 т.п.о. до около 140 т.п.о.50. The method of Claim 49, wherein the remote area is between 30 kb. to about 110 kbp, and the nucleotide insert is from about 40 kbp. up to about 140 kb 51. Способ по п. 1 или 2, где LTVEC составляет от 100 т.п.о. до 300 т.п.о., 5’ и 3’ плечи гомологии в общей сумме составляют от 30 т.п.о. до 150 т.п.о и направленная генетическая модификация включает удаление области представляющего интерес геномного локуса, при этом делеция составляет от 30 т.п.о. до 110 т.п.о., и введение нуклеотидной вставки в представляющий интерес геномного локуса, при этом вставка составляет от 40 т.п.о. до 140 т.п.о.51. The method according to claim 1 or 2, wherein the LTVEC is from 100 kb. up to 300 kb, 5 ’and 3’, the shoulders of homology total up to 30 kb. up to 150 kb and directed genetic modification includes the removal of a region of the genomic locus of interest, with a deletion of between 30 kb. up to 110 kb, and the introduction of a nucleotide insert into a genomic locus of interest, with the insertion ranging from 40 kb. up to 140 kb 52. Способ по п. 1 или 2, где LTVEC составляет от 20 т.п.о. до 250 т.п.о. в длину, при этом 5’ и 3’ плечи гомологии в общей сумме составляют от 20 т.п.о. до 200 т.п.о в длину, и где направленная генетическая модификация включает удаление области представляющего интерес геномного локуса, при этом делеция составляет от 30 т.п.о. до 110 т.п.о., и введение нуклеотидной вставки в представляющий интерес геномный локус, при этом вставка составляет от 40 т.п.о. до 140 т.п.о., и где направляющую РНК конструируют для создания двухцепочечного разрыва в области представляющего интерес геномного локуса, предназначенного для удаления.52. The method according to claim 1 or 2, where the LTVEC is from 20 kb. up to 250 kb in length, with 5 ’and 3’ shoulders of homology totaling from 20 kb. up to 200 kb in length, and where the directed genetic modification involves the removal of a region of interest of the genomic locus, with a deletion of between 30 kb. up to 110 kb, and the introduction of a nucleotide insert into a genomic locus of interest, with the insertion ranging from 40 kb. up to 140 kb, and where the guide RNA is designed to create a double-stranded break in the region of the genomic locus of interest to be deleted. 53. Способ по пп. 1 или 2, где плюрипотентная клетка представляет собой плюрипотентную клетку грызуна.53. The method according to paragraphs. 1 or 2, where the pluripotent cell is a pluripotent rodent cell. 54. Способ по п. 53, где плюрипотентная клетка грызуна представляет собой крысиную или мышиную эмбриональную стволовую (ЭС) клетку.54. The method of claim 53, wherein the pluripotent rodent cell is a rat or mouse embryonic stem (ES) cell. 55. Способ по п. 54, где плюрипотентная клетка грызуна представляет собой крысиную ЭС клетку.55. The method of claim 54, wherein the pluripotent rodent cell is a rat ES cell. 56. Способ по п. 55, где крысиную ЭС культивируют на слое питающих клеток митотически инактивированных фибробластов эмбриона мыши со средой 2i, состав которой приведен в таблице 3,56. The method according to claim 55, wherein the rat ES is cultured on a layer of feeder cells of mitotic inactivated mouse embryonic fibroblasts with medium 2i, the composition of which is shown in Table 3, где направленная генетическая модификация может быть передана через зародышевую линию.where directed genetic modification can be transmitted through the germ line. 57. Способ по п.55 или 56, дополнительно включающий57. The method according to p. 55 or 56, further comprising (a) выявление модифицированной крысиной ЭС клетки, содержащей направленную генетическую модификацию в представляющем интерес геномном локусе; (a) identifying a modified rat ES cell containing a directed genetic modification in a genomic locus of interest; (b) внесение модифицированной крысиной ЭС клетки в крысиный эмбрион-хозяин; и (b) insertion of modified rat ES cells into a host rat embryo; and (c) вынашивание крысиного эмбриона-хозяина суррогатной матерью, при этом суррогатная мать производит крысиное поколение F0, содержащее направленную генетическую модификацию в представляющем интерес геномном локусе.(c) nursing a rat host embryo by a surrogate mother, with the surrogate mother producing the F0 rat generation containing a directed genetic modification in the genomic locus of interest. 58. Способ по п.54, где плюрипотентная клетка грызуна представляет собой мышиную ЭС клетку.58. The method according to claim 54, where the pluripotent rodent cell is a mouse ES cell. 59. Способ по п.58, дополнительно включающий59. The method according to § 58, further comprising (a) выявление модифицированной мышиной ЭС клетки, содержащей направленную генетическую модификацию в представляющем интерес геномном локусе; (a) identifying a modified murine ES cell containing a directed genetic modification in a genomic locus of interest; (b) внесение модифицированной мышиной ЭС клетки в мышиный эмбрион-хозяин; и (b) insertion of modified murine ES cells into a mouse host embryo; and (c) вынашивание мышиного эмбриона-хозяина суррогатной матерью, при этом суррогатная мать производит мышиное поколение F0, содержащее направленную генетическую модификацию в представляющем интерес геномном локусе.(c) gestation of a mouse host embryo by a surrogate mother, with the surrogate mother producing a mouse F0 generation containing targeted genetic modification in a genomic locus of interest.
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